Immunogenic peptides and methods of use for treating and preventing cancer

ABSTRACT

Disclosed are immunogenic peptides, related fusion proteins, nucleic acids encoding the peptides or fusion proteins, conjugates, expression vectors, host cells, and antibodies. Also, disclosed are pharmaceutical compositions, vaccines for use in the treatment or prevention of cancer, e.g., alveolar rhabodomyosarcoma, methods of stimulating a T cell to kill a tumor cell, methods of stimulating CD4 +  and CD8 +  T cells, and methods of treating or preventing cancer are further provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/966,341, filed Dec. 13, 2010, which is a divisional of U.S. patentapplication Ser. No. 12/092,449, filed Oct. 6, 2008, now U.S. Pat. No.7,867,977, which is a National Stage of International Application No.PCT/US06/41462, filed Oct. 24, 2006, which claims priority to U.S.Patent Application No. 60/733,319, which was filed on Nov. 3, 2005. Thedisclosure of each of these related applications is incorporated hereintheir entireties.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 13,830 Byte ASCII (Text) file named“715840ST25.txt,” dated Dec. 20, 2013.

BACKGROUND OF THE INVENTION

Most tumors express mutated or inappropriately expressed, nonmutatedtumor-associated antigens (TAAs) that often contain cytotoxic Tlymphocyte (CTL) epitopes. Yet, the immune system often remainsincapable of overtaking the growth potential of the malignant cells.Many approaches have been attempted to obtain protective and therapeuticanti-tumor immunity. However, for some of these approaches, limitedsuccess was observed (Dagher et al., Med Pediatr Oncol 38: 158-164(2002); and Rodeberg et al., Cancer Immuno Immunother 54: 526-534(2005)).

The present invention seeks to overcome the aforementioned problems byproviding immunogenic peptides, dendritic cells presenting theimmunogenic peptides, and methods of treating and preventing cancer.These and other advantages of the invention, as well as additionalinventive features, will be apparent from the description of theinvention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides immunogenic peptides which bind to aMajor Histocompatibility Complex (MHC) Class I molecule, e.g., HLA-B7.Fusion proteins and conjugates comprising at least one of the inventiveimmunogenic peptides described herein are also provided by the presentinvention.

The present invention further provides nucleic acids encoding any of theinventive immunogenic peptides or fusion proteins described herein,expression vectors comprising the nucleic acids, and host cellscomprising the vectors. Isolated antibodies, or antigen binding portionsthereof, that bind to any of the inventive immunogenic peptidesdescribed herein are furthermore provided by the present invention.

Pharmaceutical compositions comprising any of the inventive immunogenicpeptides, fusion proteins, conjugates, nucleic acids, expressionvectors, host cells, or antibodies, and a pharmaceutically acceptablecarrier, are provided herein. Also, vaccines comprising any of theinventive immunogenic peptides, fusion proteins, conjugates, nucleicacids, expression vectors, or host cells are provided.

Methods of stimulating a T cell to kill a tumor cell, methods ofstimulating CD4⁺ and CD8⁺ T cells, as well as methods of treating orpreventing cancer, are further provided by the present invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 depicts the CTL lytic activity at different effector cell:targetcell (E:T) ratios. The effector cells are the CTL generated in Example 2described herein, while the target cells are C1R-B7 cells pulsed withRS10 peptide (squares) or control peptide (triangles).

FIG. 2 depicts the CTL lytic activity at different E:T ratios. Theeffector cells are the CTL generated in Example 2 described herein,while the target cells are C1R-B7 cells pulsed with RS10 peptide(squares) or control peptide (triangles). Anti-HLA-B7 antibody used at10% v/v (open circles) or 20% v/v (closed circles).

FIG. 3 depicts the CTL lytic activity at different E:T ratios. Theeffector cells are the CTL generated in Example 2 described herein,while the target cells are rhabdomyosarcoma cells expressing HLA-B7(Rh5; squares), or two control cell lines, RD (triangles) or CTR(circles), which do not express HLA-B7.

FIG. 4 depicts the fluorescence index at different concentrations ofRS10 peptide (squares) or RS10-3A mutant peptide (triangles).

FIG. 5 depicts the CTL lytic activity at different E:T ratios, whereinthe effector cells are the CTL generated in Example 2 described herein,and the target cells are C1R-B7 cells pulsed with either RS10-3A peptide(squares) or control peptide (SS1; triangles).

FIG. 6 depicts the fluorescence index at different concentrations ofRS10 peptide (squares), RS10-3A mutant peptide (triangles), RS10-5Amutant peptide (circles), or RA10-6A mutant peptide (diamonds).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to immunogenic peptides. In oneembodiment, each of the peptides has an amino acid sequence based on thePAX3-FKHR fusion protein breakpoint region. The amino acid andnucleotide sequences of the PAX3-FKHR fusion protein are known in theart (SEQ ID NOs: 1 and 2, respectively) (See, for instance, Galili etal., Nature Genetics 5: 230-235 (1993), and Shapiro et al., CancerResearch 6: 5108-5112 (1993)).

In a preferred embodiment, the present inventive immunogenic peptidecomprises the amino acid sequence SPQNSIRHNL (SEQ ID NO: 3), but doesnot consist of the amino acid sequence TIGNGLSPQNSIRHNLSL (SEQ ID NO: 4)or NPTGTIGNGLSPQNSIRHNLSLH (SEQ ID NO: 5).

In another embodiment, the present inventive immunogenic peptidecomprises the amino acid sequence SPX₁NX₂X₃RHNL (SEQ ID NO: 6), whereinX₁ is any amino acid except for Gln, X₂ is any amino acid except forSer, and X₃ is any amino acid except for Ile.

The present inventive immunogenic peptides have one or more attractiveproperties. The peptides bind to an MHC Class I molecule. The MHC ClassI molecule to which the peptide binds can be any MHC Class I moleculeknown in the art (see, for example, Janeway et al., Immunobiology: TheImmune System in Health and Disease, 4^(th) ed., Current BiologyPublications: Garland Publishing, New York, N.Y., 1999). The MHC Class Imolecule can, for example, be an MHC Class I molecule of any mammal,e.g., mouse, rat, human rabbit, etc. Suitable MHC Class I moleculesinclude, for example, HLA-A, -B, and -C molecules, such as HLA-B7, -B8,B44, -A2, -A3, -A11, -A31, and -C1. Preferably, the MHC Class I moleculeis HLA-B7. As one of ordinary skill in the art appreciates, it ispossible for the inventive immunogenic peptides to bind to more than oneMHC Class I molecule or to both an MHC Class I molecule and an MHC ClassII molecule. Immunogenic peptides having such dual- ormulti-specificities for MHC molecules are included within the scope ofthe invention.

Methods of determining whether a given peptide binds to an MHC Class Imolecule are known in the art, and include, for instance, bindingassays, such as Far Western binding assays, surface plasmon resonancebinding assays, and the binding assay as illustrated in Example 1.

Desirably, the present inventive immunogenic peptides or portionsthereof not only bind to an MHC Class I molecule, but also stimulateCD8⁺ T cells, e.g., cytotoxic T lymphocytes (CTL). By “stimulate” in thecontext of T cells is meant activating intracellular signaling pathwaysin a T cell through the antigen-specific T cell receptor (TCR) expressedon that T cell, which activation leads to one or more T cell responses,such as T cell proliferation, cytolytic activity, and cytokineproduction, e.g., IFN-γ. Preferably, the T cells are stimulated by thepresent inventive peptides to kill or lyse a target cell, which presentsthe peptide recognized by the TCR of the T cell. Desirably, the targetcell is a tumor cell. Also, in some instances, it is preferable for thepeptides to stimulate CD4⁺ T cells, in addition to CD8⁺ T cells.Stimulation of CD4⁺ T cells by the immunogenic peptide desirably aids aB cell mediated immune response, which includes the production ofantibodies.

The immunogenic peptides of the present invention can be of any length,i.e., can comprise any number of amino acids, provided that the peptidesare able to bind to an MHC Class I molecule. For example, the peptidecan be 5 to 654 amino acids long, such as 5, 6, 7, 8, 9, 10, 11, 12, 13,15, 17, 19, 20, 25, 50, 75, 100 or more amino acids in length. In apreferred embodiment, the peptide consists of 8 to 10 amino acids.

The immunogenic peptides of the present invention can comprise syntheticamino acids in place of one or more naturally-occurring amino acids.Such synthetic amino acids are known in the art, and include, forexample, aminocyclohexane carboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The present inventive immunogenic peptides can be glycosylated,amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclizedvia, e.g., a disulfide bridge, or converted into an acid addition saltand/or optionally dimerized or polymerized, or conjugated.

When the immunogenic peptides of the present invention are in the formof a salt, preferably, the peptides are in the form of apharmaceutically acceptable salt. Suitable pharmaceutically acceptableacid addition salts include those derived from mineral acids, such ashydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, andsulphuric acids, and organic acids, such as tartaric, acetic, citric,malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, andarylsulphonic acids, for example, p-toluenesulphonic acid.

The present invention also provides functional portions of theimmunogenic peptides described herein. The term “functional portion”when used in reference to an immunogenic peptide refers to any part orfragment of the immunogenic peptide of the present invention, which partor fragment retains the biological (e.g., immunogenic) activity of theimmunogenic peptide of which it is a part. Functional portionsencompass, for example, those parts of an immunogenic peptide (theparent peptide) that retain the ability to bind to an MHC Class Imolecule to a similar extent, the same extent, or to a higher extent, asthe parent peptide. In reference to the parent peptide, the functionalportion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%,90%, 95%, or more of the parent peptide. The functional portion cancomprise additional amino acids at the amino or carboxy terminus of theportion, or at both termini, which additional amino acids are not foundin the amino acid sequence of the parent peptide. Desirably, theadditional amino acids do not interfere with the biological function ofthe functional portion, e.g., binding to an MHC Class I molecule,stimulation of CD8⁺ and CD4⁺ T cells, stimulation of T cells to killtumor cells, and treatment and/or prevention of cancer.

The present invention also provides functional variants of theimmunogenic peptides described herein. The term “functional variant” asused herein refers to an immunogenic peptide having substantial orsignificant sequence identity or similarity to a parent immunogenicpeptide, which functional variant retains the biological activity of theimmunogenic peptide of which it is a variant. Functional variantsencompass, for example, those variants of the immunogenic peptide (theparent peptide) that retain the ability to bind to an MHC Class Imolecule to a similar extent, the same extent, or to a higher extent, asthe parent peptide. In reference to the parent peptide, the functionalvariant can, for instance, be at least 30%, 50%, 75%, 80%, 90%, 98% ormore identical to the parent peptide.

The functional variant can, for example, comprise the amino acidsequence of the parent immunogenic peptide with at least oneconservative amino acid substitution. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the samechemical or physical properties. For instance, the conservative aminoacid substitution can be an acidic amino acid substituted for anotheracidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar sidechain substituted for another amino acid with a nonpolar side chain(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basicamino acid substituted for another basic amino acid (Lys, Arg, etc.), anamino acid with a polar side chain substituted for another amino acidwith a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent immunogenic peptide with at least onenon-conservative amino acid substitution. In this case, it is preferablefor the non-conservative amino acid substitution to not interfere withor inhibit the biological activity of the peptide. Preferably, thenon-conservative amino acid substitution enhances the biologicalactivity of the peptide.

The immunogenic peptides can consist essentially of the specified aminoacid sequence, such other components of the peptide, e.g., other aminoacids, do not materially change the biological, e.g., immunogenic,activity of the peptide. In this regard, the present inventiveimmunogenic peptide can, for example, consist essentially of the aminoacid sequence SPQNSIRHNL (SEQ ID NO: 3). Also, for instance, theinventive peptide can consist essentially of the amino acid sequence ofSEQ ID NO: 5.

With respect to the immunogenic peptides comprising an amino acidsequence of SEQ ID NO: 5, it is preferred that each of X₁, X₂ and X₃ isindependently any small, aliphatic amino acid. Small aliphatic aminoacids are known in the art and include, for example, Ser, Thr, or Ala.In a more preferred embodiment, X₁ is Ser, Thr, or Ala; X₂ is Thr orAla; and X₃ is Ser, Thr, or Ala. In an even more preferred embodiment,the peptide comprises an amino acid sequence selected from the groupconsisting of: SPANSIRHNL (SEQ ID NO: 7); SPQNAIRHNL (SEQ ID NO: 8); andSPQNSARHNL (SEQ ID NO: 9).

With respect to the immunogenic peptide comprising the amino acidsequence of SEQ ID NO: 3, it is preferred that the peptide consists orconsists essentially of the amino acid sequence SPQNSIRHNL (SEQ ID NO:3). Also, with respect to the immunogenic peptides of SEQ ID NO: 3, itis preferred that the peptides are isolated and/or purified.

The immunogenic peptides of the present invention can be obtained bymethods known in the art. Suitable methods of de novo synthesizingpeptides are described herein and in references, such as Chan et al.,Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford,United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R.,Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwoood et al., OxfordUniversity Press, Oxford, United Kingdom, 2000; and U.S. Pat. No.5,449,752. Also, peptides can be recombinantly produced using thenucleic acids described herein using standard recombinant methods. See,for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, NY, 1994. Further, some ofthe immunogenic peptide can be isolated and/or purified from a source,such as a plant, a bacterium, a mammal, e.g., a rat, a human, etc.Methods of isolation and purification are well-known in the art.Alternatively, the peptides described herein can be commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), and Multiple Peptide Systems(San Diego, Calif.). In this respect, the immunogenic peptides of thepresent invention can be synthetic, recombinant, isolated, and/orpurified.

The present invention further provides a fusion protein comprising atleast one of the immunogenic peptides (including functional portions andvariants thereof) described herein and an MHC Class I molecule, or aportion thereof. The MHC Class I molecule can be any of the MHC Class Imolecules known in the art, such as any of those described herein.Preferably, the MHC Class I molecule is HLA-B7. The portion of the MHCClass I molecule can be any part of the MHC Class I molecule.Preferably, the portion comprises the peptide binding portion of the MHCClass I molecule. More preferably, the portion comprises the peptidebinding portion and the T cell receptor binding portion of the MHC ClassI molecule. Such portions of MHC Class I molecules are known in the art.

The fusion protein can comprise one or more copies of the immunogenicpeptide and/or one or more copies of the MHC Class I molecule or partthereof. For instance, the fusion protein can comprise 1, 2, 3, 4, 5, ormore copies of the immunogenic peptide and/or of the MHC Class Imolecule or part thereof. Suitable methods of making fusion proteins areknown in the art, and include, for example, recombinant methods. See,for instance, Choi et al., Mol Biotechnol 31: 193-202 (2005).

The present invention further provides conjugates, e.g., bioconjugates,comprising any of the immunogenic peptides (including any of thefunctional portions or variants thereof). Conjugates, as well as methodsof synthesizing conjugates of peptides in general, are known in the art(See, for instance, Hudecz, F., Methods Mol Biol 298: 209-223 (2005) andKirin et al., Inorg Chem 44(15): 5405-5415 (2005)).

The present invention provides a nucleic acid comprising a nucleotidesequence encoding any of the immunogenic peptides, functional portionsor variants thereof, or fusion proteins thereof, described herein. By“nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. It is generally preferred that thenucleic acid does not comprise any insertions, deletions, inversions,and/or substitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the present invention are recombinant.As used herein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., supra, and Ausubel et al., supra. For example,a nucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, one or more ofthe nucleic acids of the present invention can be purchased fromcompanies, such as Macromolecular Resources (Fort Collins, Colo.) andSynthegen (Houston, Tex.).

The nucleic acids of the present invention can be incorporated into anexpression vector. In this regard, the present invention providesexpression vectors comprising any of the nucleic acids of the presentinvention. For purposes herein, the term “expression vector” means agenetically-modified oligonucleotide (i.e., polynucleotide) constructthat permits the expression of a protein or a peptide by a host cell,when the construct comprises a nucleotide sequence encoding the proteinor peptide, and the vector is contacted with the cell under conditionssufficient to have the protein expressed within the cell. The vectors ofthe present invention are not naturally-occurring as a whole. However,parts of the vectors can be naturally-occurring. The present inventiveexpression vectors can comprise any type of nucleotides, including, butnot limited to DNA and RNA, which can be single-stranded ordouble-stranded, synthesized or obtained in part from natural sources,and which can contain natural, non-natural or altered nucleotides. Theexpression vectors can comprise naturally-occurring,non-naturally-occurring internucleotide linkages, or both types oflinkages. Preferably, the non-naturally occurring or altered nucleotidesor internucleotide linkages does not hinder in any way the transcriptionor replication of the vector.

The expression vector of the present invention can be any suitableexpression vector, and can be used to transform or transfect anysuitable host. Suitable vectors include those designed for propagationand expansion or for expression or both, such as plasmids and viruses.The vector can be selected from the group consisting of the pUC series(Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla,Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series(Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, PaloAlto, Calif.). Bacteriophage vectors, such as λGT10, λGT11, λZapII(Stratagene), λEMBL4, and λNM1149, also can be used. Examples of plantexpression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19(Clontech). Examples of animal expression vectors include pEUK-Cl, pMAMand pMAMneo (Clontech).

The expression vectors of the present invention can be prepared usingstandard recombinant DNA techniques described in, for example, Sambrooket al., supra, and Ausubel et al., supra. Constructs of expressionvectors, which are circular or linear, can be prepared to contain areplication system functional in a prokaryotic or eukaryotic host cell.Replication systems can be derived, e.g., from ColEl, 2μ plasmid, λ,SV40, bovine papilloma virus, and the like.

Desirably, the expression vector comprises regulatory sequences, such astranscription and translation initiation and termination codons, whichare specific to the type of host (e.g., bacterium, fungus, plant, oranimal) into which the vector is to be introduced, as appropriate andtaking into consideration whether the vector is DNA- or RNA-based.

The expression vector can include one or more marker genes, which allowfor selection of transformed or transfected hosts. Marker genes includebiocide resistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike. Suitable marker genes for the present inventive expression vectorsinclude, for instance, neomycin/G418 resistance genes, hygromycinresistance genes, histidinol resistance genes, tetracycline resistancegenes, and ampicillin resistance genes.

The expression vector can comprise a native or nonnative promoteroperably linked to the nucleic acid encoding the protein. The selectionof promoters, e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the ordinary skill of the artisan.Similarly, the combining of a nucleic acid with a promoter is alsowithin the skill of the artisan. The promoter can be a non-viralpromoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

The present inventive expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, theexpression vectors can be made for constitutive expression or forinducible expression. Further, the expression vectors can be made toinclude a suicide gene.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene can be a genethat confers sensitivity to an agent, e.g., a drug, upon the cell inwhich the gene is expressed, and causes the cell to die when the cell iscontacted with or exposed to the agent. Suicide genes are known in theart (see, for example, Suicide Gene Therapy: Methods and Reviews,Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeuticsat the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press,2004) and include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase,and nitroreductase.

The present invention further provides a host cell comprising any of theexpression vectors described herein. As used herein, the term “hostcell” refers to any type of cell that can contain the present inventiveexpression vector. The host cell can be a eukaryotic cell, e.g., plant,animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria orprotozoa. The cell can be a cultured cell or a primary cell, i.e.,isolated directly from an organism, e.g., a human. The cell can be anadherent cell or a suspended cell, i.e., a cell that grows insuspension. Suitable host cells are known in the art and include, forinstance, DH5α E. coli cells, Chinese hamster ovarian cells, monkey VEROcells, COS cells, HEK293 cells, and the like. For purposes of amplifyingor replicating the recombinant expression vector, the host cell ispreferably a prokaryotic cell, e.g., a DH5α cell. For purposes ofproducing a recombinant protein, the host cell is preferably a mammaliancell. Most preferably, the host cell is a human cell. While the hostcell can be of any cell type, can originate from any type of tissue, andcan be of any developmental stage, the host cell preferably is anantigen presenting cell, such as, for instance, a dendritic cell, amacrophage, or a B cell. Preferably, the host cell is a dendritic cell.

The present invention further provides an antibody, or an antigenbinding portion thereof, that binds to any of the immunogenic peptidesdescribed herein. The antibody can be any type of immunoglobulin that isknown in the art. For instance, the antibody can be of any isotype,e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal orpolyclonal. The antibody can be a naturally-occurring antibody, e.g., anantibody isolated and/or purified from a mammal, e.g., mouse, rabbit,goat, horse, chicken, hamster, human, etc. Alternatively, the antibodycan be a genetically-engineered antibody, e.g., a humanized antibody ora chimeric antibody. The antibody can be in monomeric or polymeric form.Also, the antibody can have any level of affinity or avidity for thepeptide of the present invention. Desirably, the antibody is specificfor the peptide, such that there is minimal cross-reaction with otherpeptides or proteins.

Methods of testing antibodies for the ability to bind to any of theimmunogenic peptides are known in the art an include anyantibody-antigen binding assay, such as, for example, radioimmunoassay(RIA), ELISA, Western blot, immunoprecipitation, and competitiveinhibition assays (see, e.g., Janeway et al., supra, and U.S. PatentApplication Publication No. 2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Köhler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing, New York,N.Y. (2001)). Alternatively, other methods, such as EBV-hybridomamethods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984),and Roder et al., Methods Enzymol., 121, 140-67 (1986)), andbacteriophage vector expression systems (see, e.g., Huse et al.,Science, 246, 1275-81 (1989)) are known in the art. Further, methods ofproducing antibodies in non-human animals are described in, e.g., U.S.Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication No. 2002/0197266 A1).

Phage display furthermore can be used to generate the antibody of thepresent invention. In this regard, phage libraries encodingantigen-binding variable (V) domains of antibodies can be generatedusing standard molecular biology and recombinant DNA techniques (see,e.g., Sambrook et al. (eds.), Molecular Cloning, A Laboratory Manual,3^(rd) Edition, Cold Spring Harbor Laboratory Press, New York (2001)).Phage encoding a variable region with the desired specificity areselected for specific binding to the desired antigen, and a complete orpartial antibody is reconstituted comprising the selected variabledomain. Nucleic acid sequences encoding the reconstituted antibody areintroduced into a suitable cell line, such as a myeloma cell used forhybridoma production, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol.,235, 959-973 (1994).

The present inventive invention also provides antigen binding portionsof any of the antibodies described herein. The antigen binding portioncan be any portion that has at least one antigen binding site, such asFab, F(ab′)₂, dsFv, sFv, diabodies, and triabodies.

A single-chain variable region fragment (sFv) antibody fragment, whichconsists of a truncated Fab fragment comprising the variable (V) domainof an antibody heavy chain linked to a V domain of a light antibodychain via a synthetic peptide, can be generated using routinerecombinant DNA technology techniques (see, e.g., Janeway et al.,supra). Similarly, disulfide-stabilized variable region fragments (dsFv)can be prepared by recombinant DNA technology (see, e.g., Reiter et al.,Protein Engineering, 7, 697-704 (1994)). Antibody fragments of thepresent invention, however, are not limited to these exemplary types ofantibody fragments.

The present inventive immunogenic peptides, fusion proteins, conjugates,nucleic acids, expression vectors, host cells, and antibodies, can beisolated and/or purified. The term “isolated” as used herein meanshaving been removed from its natural environment. The term “purified” asused herein means having been increased in purity, wherein “purity” is arelative term, and not to be necessarily construed as absolute purity.For example, the purity can be at least 50%, can be greater than 60%,70% or 80%, or can be 100%.

The present inventive immunogenic peptides (including functionalportions and variants thereof), fusion proteins, conjugates, nucleicacids, expression vectors, host cells, and antibodies (including antigenbinding portions thereof), all of which are collectively referred to as“immunogenic materials” hereinafter, can be formulated into acomposition, such as a pharmaceutical composition. In this regard, thepresent invention provides a pharmaceutical composition comprising anyof the immunogenic peptides, fusion proteins, conjugates, nucleic acids,expression vectors, host cells, and antibodies, and a pharmaceuticallyacceptable carrier. The present inventive pharmaceutical compositionscontaining any of the immunogenic materials can comprise more than oneimmunogenic material, e.g., a peptide and a nucleic acid, or two or moredifferent peptides. Alternatively, the pharmaceutical composition cancomprise an immunogenic material in combination with anotherpharmaceutically active agents or drugs, such as a chemotherapeuticagents e.g., asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with theactive compound(s), and by the route of administration. Thepharmaceutically acceptable carriers described herein, for example,vehicles, adjuvants, excipients, and diluents, are well-known to thoseskilled in the art and are readily available to the public. It ispreferred that the pharmaceutically acceptable carrier be one which ischemically inert to the active agent(s) and one which has no detrimentalside effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularimmunogenic material, as well as by the particular method used toadminister the immunogenic material. Accordingly, there are a variety ofsuitable formulations of the pharmaceutical composition of the presentinvention. The following formulations for oral, aerosol, parenteral,subcutaneous, intravenous, intramuscular, intraarterial, intrathecal,interperitoneal, rectal, and vaginal administration are exemplary andare in no way limiting. More than one route can be used to administerthe immunogenic materials, and in certain instances, a particular routecan provide a more immediate and more effective response than anotherroute.

Topical formulations are well-known to those of skill in the art. Suchformulations are particularly suitable in the context of the presentinvention for application to the skin.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the immunogenic materialdissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the immunogenic material in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the immunogenic material in an inert base, such as gelatinand glycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to, such excipients as are known in the art.

The immunogenic material, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. They also may be formulated as pharmaceuticalsfor non-pressured preparations, such as in a nebulizer or an atomizer.Such spray formulations also may be used to spray mucosa.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The immunogenic material can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain from about 0.5% toabout 25% by weight of the immunogenic material in solution.Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene glycol sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Injectable formulations are in accordance with the present invention.The requirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)). Preferably, when administering cells, e.g., dendritic cells,the cells are administered via injection.

Additionally, the immunogenic materials, or compositions comprising suchimmunogenic materials, can be made into suppositories by mixing with avariety of bases, such as emulsifying bases or water-soluble bases.Formulations suitable for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the immunogenicmaterials of the present invention can be formulated as inclusioncomplexes, such as cyclodextrin inclusion complexes, or liposomes.

For purposes of the present invention, the amount or dose of theimmunogenic material administered should be sufficient to effect, e.g.,a therapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the immunogenic materialshould be sufficient to bind to an MHC Class I molecule, stimulate tumorcell killing, stimulate CD4⁺ and CD8⁺ T cells, or treat or preventcancer in a period of from about 2 hours or longer, e.g., 12 to 24 ormore hours, from the time of administration. In certain embodiments, thetime period could be even longer. The dose will be determined by theefficacy of the particular immunogenic material and the condition of theanimal (e.g., human), as well as the body weight of the animal (e.g.,human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes of the present invention, an assay, which comprisescomparing the extent to which CD8⁺ T cells are stimulated to kill tumorcells upon administration of a given dose of a immunogenic material to amammal among a set of mammals of which is each given a different dose ofthe immunogenic material, could be used to determine a starting dose tobe administered to a mammal. The extent to which the tumor cells arekilled upon administration of a certain dose can be assayed by methodsknown in the art, including, for instance, the method described hereinas Example 3.

The dose of the immunogenic material also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular immunogenic material.Typically, the attending physician will decide the dosage of theimmunogenic material with which to treat each individual patient, takinginto consideration a variety of factors, such as age, body weight,general health, diet, sex, immunogenic material to be administered,route of administration, and the severity of the condition beingtreated. By way of example and not intending to limit the presentinvention, the dose of the immunogenic material can be about 0.001 toabout 1000 mg/kg body weight of the subject being treated/day, fromabout 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1mg/kg body weight/day.

One of ordinary skill in the art will readily appreciate that theimmunogenic materials of the present invention can be modified in anynumber of ways, such that the therapeutic or prophylactic efficacy ofthe immunogenic materials is increased through the modification. Forinstance, the immunogenic materials can be conjugated either directly orindirectly through a linker to a targeting moiety. The practice ofconjugating compounds, e.g., immunogenic materials, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J. DrugTargeting 3: 111 (1995) and U.S. Pat. No. 5,087,616. The term “targetingmoiety” as used herein, refers to any molecule or agent thatspecifically recognizes and binds to a cell-surface receptor, such thatthe targeting moiety directs the delivery of the immunogenic materialsto a population of cells on which surface the receptor is expressed.Targeting moieties include, but are not limited to, antibodies, orfragments thereof, peptides, hormones, growth factors, cytokines, andany other natural or non-natural ligands, which bind to cell surfacereceptors (e.g., Epithelial Growth Factor Receptor (EGFR), T-cellreceptor (TCR), B-cell receptor (BCR), CD28, Platelet-derived GrowthFactor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).The term “linker” as used herein, refers to any agent or molecule thatbridges the immunogenic materials to the targeting moiety. One ofordinary skill in the art recognizes that sites on the immunogenicmaterials, which are not necessary for the function of the immunogenicmaterials, are ideal sites for attaching a linker and/or a targetingmoiety, provided that the linker and/or targeting moiety, once attachedto the immunogenic materials, do(es) not interfere with the function ofthe immunogenic materials, i.e., the ability to bind to an MHC Class Imolecule, to stimulate CD4⁺ and CD8⁺ T cells, to stimulate the killingof tumor cells, or to treat or prevent cancer.

Alternatively, the immunogenic materials can be modified into a depotform, such that the manner in which the immunogenic materials isreleased into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of immunogenic materials can be, forexample, an implantable composition comprising the immunogenic materialsand a porous or non-porous material, such as a polymer, wherein theimmunogenic materials is encapsulated by or diffused throughout thematerial and/or degradation of the non-porous material. The depot isthen implanted into the desired location within the body and theimmunogenic materials are released from the implant at a predeterminedrate.

The present inventive immunogenic peptides (including functionalportions and variants thereof), fusion proteins, conjugates, nucleicacids, expression vectors, and host cells can be made as part of avaccine. As such, the present invention also provides a vaccinecomprising any of the immunogenic peptides, fusion proteins, nucleicacids, expression vectors, and host cells described herein. The term“vaccine” as used herein means any substance that causes activation ofan animal's immune system without causing actual disease. The vaccinesof the present invention comprise an immunogen that induces an immuneresponse directed against a tumor antigen, e.g., PAX3-FKHR. Theimmunogen of the present inventive vaccines is any of the immunogenicpeptides, fusion proteins, nucleic acids, expression vectors, and hostcells described herein.

In one embodiment, the vaccine is an expression vector encoding aPAX3-FKHR peptide. To provide a vaccine to an individual, a nucleotidesequence which encodes for the PAX3-FKHR peptide is inserted into aexpression vector, as described above, and introduced into the mammal tobe immunized. Examples of vectors that may be used in the aforementionedvaccines include, but are not limited to, defective retroviral vectors,adenoviral vectors vaccinia viral vectors, fowl pox viral vectors, orother viral vectors (Mulligan, R. C., (1993) Science 260:926-932). Theviral vectors carrying the PAX3-FKHR nucleic acid can be introduced intoa mammal either prior to any evidence of the disease, e.g., cancer, orto mediate regression of the disease in a mammal afflicted with thedisease. Examples of methods for administering the viral vector into themammals include, but are not limited to, exposure of cells to the virusex vivo, or injection of the retrovirus or a producer cell line of thevirus into the affected tissue or intravenous administration of thevirus. Alternatively, the viral vector carrying all or part of thePAX3-FKHR nucleic acid sequence may be administered locally by directinjection or topical application in a pharmaceutically acceptablecarrier.

The quantity of viral vector carrying the PAX3-FKHR nucleic acidsequence to be administered is based on the titer of virus particles. Apreferred range of the immunogen to be administered may be about 10⁶ toabout 10¹¹ virus particles per mammal, preferably a human. Afterimmunization, the efficacy of the vaccine can be assessed by productionof antibodies or immune cells that recognize the antigen (PAX3-FKHR), asassessed by specific lytic activity or specific cytokine production orby regression of the disease. One skilled in the art knows theconventional methods to assess the aforementioned parameters. If themammal to be immunized is already afflicted with the disease, thevaccine can be administered in conjunction with other therapeutictreatments. Examples of other therapeutic treatments includes, but arenot limited to, adoptive T cell immunotherapy, coadministration ofcytokines or other therapeutic drugs for the disease.

Alternatively, the PAX3-FKHR peptides may be administered as a vaccinein a pharmaceutically acceptable carrier. Ranges of PAX3-FKHR peptidethat may be administered are about 0.001 to about 100 mg per patient,preferred doses are about 0.01 to about 100 mg per patient. Immunizationis repeated as necessary, until a sufficient titer of anti-immunogenantibody or immune cells has been obtained.

In yet another alternative embodiment, mammalian cells expressing thePAX3-FKHR antigen can be administered to mammals and serve as a vaccine.Examples of mammalian cells include, but are not limited to, primarymammalian cultures or continuous mammalian cultures, COS cells, NIH3T3,or 293 cells (ATCC #CRL 1573). Examples of how the cells expressingPAX3-FKHR antigens can be administered include, but not limited to,intravenous, intraperitoneal or intralesional administration.

In yet another embodiment of this invention, PAX3-FKHR peptides, may beexposed to dendritic cells cultured in vitro. The cultured dendriticcells provide a means of producing T-cell dependent antigens comprisedof dendritic cell modified antigen or dendritic cells pulsed withantigen, in which the antigen is processed and expressed on the antigenactivated dendritic cell. The PAX3-FKHR antigen-activated dendriticcells or processed dendritic cell antigens may be used as immunogens forvaccines or for the treatment of cancer. The dendritic cells should beexposed to antigen for sufficient time to allow the antigens to beinternalized and presented on the dendritic cells surface. The resultingdendritic cells or the dendritic cell process antigens can than beadministered to an individual in need of therapy. Such methods aredescribed in Steinman et al. (WO 93/08185) and in Banchereau et al. (EPApplication 0563485A1) which are incorporated herein by reference.

Vaccination can be conducted by conventional methods. For example, theimmunogen can be used in a suitable diluent, such as saline or water, ora complete or incomplete adjuvant. Further, the immunogen can be boundor unbound to a carrier to make the peptide immunogenic. Examples ofsuch carrier molecules include but are not limited to bovine serumalbumin (BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and thelike. The immunogen also can be coupled with a lipoprotein oradministered in liposomal form or with adjuvants. The immunogen can beadministered by any route appropriate for antibody production such asintravenous, intraperitoneal, intramuscular, subcutaneous, and the like.The immunogen may be administered once or at periodic intervals until asignificant titer of anti-PAX3-FKHR immune cells or anti-PAX3-FKHRantibody is produced. The presence of anti-PAX3-FKHR immune cells may beassessed by measuring the frequency of precursor CTL against PAX3-FKHRantigen prior to and after immunization by a CTL precursor analysisassay (Coulie, P. et al., (1992) Internat J Can 50:289-297). Theantibody may be detected in the serum using standard immunoassays knownin the art.

The vaccine formulations may be evaluated first in animal models,initially rodents, and in non-human primates and finally in humans. Thesafety of the immunization procedures is determined by looking for theeffect of immunization on the general health of the immunized animal(weight change, fever, appetite behavior etc.) and looking forpathological changes on autopsies. After initial testing in animals,diseased patients can be tested. Conventional methods would be used toevaluate the immune response of the patient to determine the efficiencyof the vaccine.

The vaccine may be used either prophylactically or therapeutically. Whenprovided prophylactically, the vaccine is provided in advance of anyevidence of disease, e.g., cancer. The prophylactic administration ofthe vaccine should serve to prevent or attenuate the disease in amammal. In a preferred embodiment, mammals, preferably humans, at highrisk for the disease are prophylactically treated with the vaccines ofthe present invention. Examples of such mammals include, but are notlimited to, humans with a family history of the disease or humanspreviously afflicted with the disease and therefore at risk forre-occurrence. When provided therapeutically, the vaccine is provided toenhance the patient's own immune response to the antigen, e.g., tumorantigen, present in the patient.

It is contemplated that the present inventive pharmaceuticalcompositions and vaccines can be used in methods of treating orpreventing cancer. Without being bound to any particular theory, it isbelieved that the present inventive peptides bind to a MHC Class Imolecule, e.g., HLA-B7, and to a T cell receptor, such that thecorresponding T cell is stimulated to create an immune response, e.g., acellular immune response. More particularly, the T cells having the TCR,which binds to the peptides of the present invention, once bound to thepeptide are stimulated to lyse and/or kill the target cell, e.g., atumor cell. In this respect, the present invention provides a method ofstimulating a T cell to kill a tumor cell. The method comprisescontacting a T cell with a cell presenting on its surface any of theimmunogenic peptides described herein and with a tumor cell in a mannereffective for the T cell to kill the tumor cell.

The tumor cell lysed by the T cell can be any type of tumor cell, suchas, for instance, a tumor cell from a benign tumor or a cancerous tumor.The tumor cell can be a tumor cell from any of the following cancers:alveolar rhabdomyosarcoma, breast cancer, prostate cancer, lung cancer,colon cancer, rectal cancer, urinary bladder cancer, non-Hodgkinlymphoma, melanoma, renal cancer, pancreatic cancer, cancer of the oralcavity, pharynx cancer, ovarian cancer, thyroid cancer, stomach cancer,brain cancer, multiple myeloma, esophageal cancer, liver cancer,cervical cancer, larynx cancer, cancer of the intrahepatic bile duct,acute myeloid leukemia, soft tissue cancer, small intestine cancer,testicular cancer, chronic lymphocytic leukemia, Hodgkin lymphoma,chronic myeloid cancer, acute lymphocytic cancer, cancer of the anus,anal canal, or anorectum, cancer of the vulva, cancer of the neck,gallbladder, or pleura, malignant mesothelioma, bone cancer, cancer ofthe joints, hypopharynx cancer, cancer of the eye, cancer of the nose,nasal cavity, or middle ear, nasopharynx cancer, ureter cancer,peritoneum, omentum, and mesentery cancer, or gastrointestinal carcinoidtumor. Preferably, the tumor cell is an alveolar rhabodomyosarcoma cell.

With respect to the present inventive method of stimulating a T cell tokill a tumor cell, the T cell can be contacted with the tumor cell andthe peptide-presenting cell simultaneously or sequentially. Forinstance, the T cell can be contacted with the peptide-presenting cellbefore being contacted with the tumor cell. Alternatively, the T cellcan be contacted with the tumor cell and the peptide-presenting cell atthe same time.

Also, the T cell can be contacted with the peptide-presenting cell invitro, in vivo or ex vivo. For example, the T cell can be contacted withthe peptide-presenting cell in vitro or ex vivo and then subsequentlycontacted with the tumor cell in vivo. Alternatively, the T cell can becontacted with the peptide-presenting cell in vivo and then subsequentlycontacted with the tumor cell in vivo, or can be contacted with thepeptide-presenting cell and the tumor cell in vivo and simultaneously.

In a preferred embodiment, the method provides for the killing ofmultiple tumor cells in a manner effective to treat cancer in a mammal.In this regard, the present invention provides a method of treatingcancer. Preferably, the T cells are autologous to the mammal beingtreated.

Further, without being bound to any particular theory, some of thepresent inventive peptides can also be able to stimulate CD4⁺ T cells,such that the T cells help B cells to produce antibodies against thepeptides. In this regard, the peptides desirably stimulate a humoralimmune response, in addition to the cellular immune response mediatedthrough the CD8⁺ T cells. Accordingly, the present invention alsoprovides methods of stimulating CD4⁺ and CD8⁺ T cells. The methodcomprises contacting a CD4⁺ and a CD8⁺ T cell with any of the dendriticcells described herein.

With respect to the present inventive method of stimulating a CD4⁺ Tcell and a CD8⁺ T cell, the T cell can be contacted with the CD4⁺ T celland a CD8⁺ T cell simultaneously or sequentially. For instance, the CD4⁺T cell can be contacted with the dendritic cell at the same or differenttime as the time that the CD8⁺ T cell is contacted with the dendriticcell.

Also, either of the CD4⁺ T cell and the CD8⁺ T cell can be contactedwith the dendritic cell in vitro, in vivo or ex vivo. For example, theCD8⁺ T cell can be contacted with the dendritic cell in vitro or exvivo. Alternatively, the T cell can be contacted with the dendritic cellin vivo.

In a preferred embodiment, the T cells, e.g., the CD8⁺ T cells, arecontacted with a tumor cell after being contacted with the dendriticcell, such that the T cell lyses and kills the tumor cell.

In a more preferred embodiment, the method provides for the killing ofmultiple tumor cells in a manner effective to treat cancer in a mammal.In this regard, the present invention provides a method of treatingcancer. Preferably, the T cells are autologous to the mammal beingtreated.

A method of treating or preventing cancer in a mammal is furtherprovided herein. The method comprises administering to the mammal any ofthe pharmaceutical compositions or vaccines described herein in anamount effective to treat or prevent cancer in the mammal.

With respect to the methods of treating or preventing cancer, the cancercan be any cancer, such as any of those described herein. Preferably,the cancer is alveolar rhabdomyosarcoma.

With respect to the methods of treating or preventing cancer, thepharmaceutical compositions or vaccines can be administered by anymethod known in the art, including any of the routes described herein.As one of ordinary skill in the art recognizes, some pharmaceuticalcompositions and vaccines are more amenable to certain routes thanothers. For example, it is preferable for the pharmaceuticalcompositions and vaccines comprising host cells to be administeredthrough injection, as opposed to orally or transdermally. The routeappropriate for the particular pharmaceutical composition or vaccine caneasily be determined by one of ordinary skill in the art.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the presentinventive methods can provide any amount of any level of treatment orprevention of cancer in a mammal.

As used herein, the term “mammal” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES

The following cell lines and peptides are used in the examples describedherein below.

C1R.B7, a specific transfectant of the B lymphoblastoid C1R cell line,which, in native form, expresses no endogenous HLA-A or HLA-B geneproducts (Storkus et al., Proc. Natl. Acad. Sci., 86, 2361-2364 (1999)),is a gift of William Biddison (National Institute of NeurologicalDisorders and Stroke, NIH, Bethesda, Md.). T2.B7 is a specifictransfectant of the hybrid B and T lymphoblastoid T2 cell line, which isdeficient in TAP1 and TAP2 gene expression (Salter et al.,Immunogenetics 21: 235-246 (1985); and Spies and DeMars, Nature 351:323-324 (1991)), and which is a gift of Peter Cresswell (YaleUniversity, New Haven, Conn.). The Rh5 alveolar rhabdomyosarcoma cellline is kindly provided by Dr. P. Houghton (St Jude's Children'sResearch Hospital). The RD and CTR embryonal rhabdomyosarcoma cell linesare obtained from the American Type Culture Collection and Dr. M. Tsokos(National Cancer Institute), respectively. Cell lines are maintained inculture medium with heat inactivated fetal calf serum (FCS) (10% v/v).Culture medium consists of RPMI1640 medium (Cellgro, Bethesda, Md.)containing L-glutamine (2 mM), penicillin (100 IU/ml), streptomycin (100μg/ml), nonessential amino acids (10 μl/ml), sodium-pyruvate (1.0 mM),gentamicm (25 μg/ml), and 2-mercaptoethanol (50 μM).

Full-length peptides are purchased from Peptide Technologies Corp.(Gaithersburg, Md.) and from Multiple Peptide Systems (San Diego,Calif.) at >95% purity and are single peaks by reverse-phasehigh-performance liquid chromatography. Optimal epitopes are synthesizedon an automated peptide synthesizer (Symphony Multiplex; ProteinTechnologies, Phoenix, Ariz.) using 9-fluoroenylmethyloxycarbonylchemistry (Stewart et al., Solid Phase Peptide Synthesis, 2″ ed.,Rockford, Ill., Pierce Chemical Company, 1984). The peptides are cleavedfrom the resin with trifluoroacetic acid. Purification to single peaksis achieved using reverse-phase high-performance liquid chromatographyon bondapack reverse-phase CIS columns (Waters Associates, Milford,Mass.).

Example 1

This example demonstrates the binding of a PAX3-FKHR peptide epitope toa MHC Class I molecule.

A chromosomal translocation-generated fusion protein breakpoint peptide,RS10 (SPQNSIRHNL), is selected on the basis of predicted potentialbinding to HLA-B7 (Rammensee et al., Immunogenetics 41: 178-228 (1995)).Peptide binding to HLA-B7 is assessed by using stabilization of HLA-B7molecules on the surface of T2-B7 cells that lack the TAP transporterand therefore express only short-lived empty HLA-B7 molecules unless apeptide is present that can bind and stabilize them. These assays aredescribed in (Stuber et al., Eur. J. Immunol., 22, 2697-2703 (1992),Nijman et al., Eur. J. Immunol., 23, 1215-1219 (1993), Zeh et al., Hum.Immunol., 39, 79-86 (1994), and Smith et al., Internat. Immunol., 9,1085-1093 (1997)). Cells of the TAP1/TAP2-deficient T2 cell line (Salteret al., Immunogenetics, 21, 235-246 (1985), and Spies et al., Nature,351, 323-324 (1991)) transfected with the HLA-B7 gene are suspended inculture medium containing heat inactivated FCS (2.5% v/v) and added to96-well round-bottomed plates at 2×10⁵ cells/well. Humanβ2-microglobulin (Sigma Chemical Co.) is also added at 20 μg/well. Whereappropriate, peptide is added to the desired concentration. The cellsare then incubated overnight at 37° C. in 5% CO₂, followed by washingwith PBS containing NaN₃ (0.5% w/v and FCS 2% v/v). Next, cells areincubated on ice for 30 min in the presence of primary anti-HLA-B7specific antibody (BB7.1 hybridoma culture supernatant; ATCC), followedby washing and incubation for 30 min in goat anti-mouse immunoglobulinFITC (Becton Dickinson). Analysis is performed by flow cytometry.

Conventional mAb staining is conducted in PBS containing 0.01% sodiumazide on ice. Cells are labeled with FITC- or PE-conjugated mAbsobtained from BD PharMingen (San Diego, Calif.). For each staining ofinterest, the appropriate isotype-matched control is included. Allreagents are used at optimal concentration as determined experimentally.Flow cytometric analysis is performed with a FACScan (BD Biosciences,Mountain View, Calif.). Data are collected on 5000-10,000 viable cellevents and analyzed with CellQuest software.

Based on these binding assays, the RS10 peptide is found to bind HLA-B7in a binding assay (data not shown).

This example demonstrated that the RS10 peptide binds to an MHC Class Imolecule.

Example 2

This example demonstrates a method of making dendritic cells pulsed withthe present inventive peptides and T cells specific for the peptides ofthe present invention.

To determine whether this peptide could elicit human CTL, dendriticcells presenting the RS10 peptide are generated. Specifically,leukopheresed human mononuclear cells are elutriated from an HLA-B7⁺normal healthy blood donor to separate a monocyte and a lymphocytefraction. The monocyte fraction is converted into dendritic cells bygrowth in GM-CSF and IL-4 and maturation with CD40L. Specifically,elutriated monocytes and lymphocytes are obtained from apheresedHLA-B7-positive subjects from the NIH normal donor pool. Monocytes arecultured for 7 days in 75 cm² culture flasks (Costar Corporation) at3.5×10⁶ cells/ml in culture medium containing heat inactivatedautologous plasma (10% v/v), h-IL-4 (800 U/ml; R&D) and h-GMCSF (50ng/ml; Immunex) at 37° C. in an atmosphere of 5% CO₂ in air. At day 3,the cells are re-fed by removing 5 ml of the medium from the cultureflasks and adding back 5 ml of fresh culture medium supplemented withcytokines (h-IL4: 400 U/ml; h-GMCSF: 25 ng/ml). At day 4, CD40 ligandtrimer (Immunex Corp., now Amgen, Seattle) is added at 1 μg/ml (Cella etal., J. Exp. Med., 184, 747-752, (1996)). Cells are harvested on day 6and stained for CD14, CD19, CD56, CD80, CD83, CD86, and HLA-DR antigens.The staining is then quantified by flow cytometry as described above.

The DCs are found to be strongly positive for CD80 and CD86costimulatory molecules, HLA-DR, and the maturation marker CD83, but arenegative for CD14, CD19, and CD56 (data not shown).

The DCs are then pulsed with the RS10 peptide and used to stimulateautologous lymphocytes from the same apheresis to generate a specificCTL line. Specifically, lymphocytes are suspended in culture mediumcontaining heat inactivated autologous plasma (10% v/v)) and plated in a24-well plate at 4×10⁶ cells/well. Autologous dendritic cells are pulsedwith RS10 peptide (10 μM) in culture medium for 4 hours. Next, thedendritic cells are irradiated with 3000 rads and added to thelymphocytes at 4×10⁵ cells/well. The next day (day 1), cultures aresupplemented with hIL-2 (12.5 U/ml), hIL-7 (2400 U/ml), hIL-1β (150U/ml) and hIL-12 (+1 ng/ml). At day 7, the cells are harvested, washed,plated in a 24-well plate at 1.5×10⁶ cells/well, and restimulated withirradiated RS10 pulsed autologous DCs (1.5×10⁵ cells/well). At day 8,the cells are supplemented with IL-2 (12.5 U/ml) and hIL-7 (2400 U/ml).Restimulations are done weekly using the same conditions. Cultures arechecked every week for relative CD4, CD8, and CD56 expression and, ifnecessary, depleted of CD4⁺ or CD56⁺ cells by magnetic cell sortingusing Magnetic Micro Beads (Miltenyi Biotec Midi-MACS).

The line is found to express CD8, but not CD4 and CD56 (data not shown).

This example demonstrated the generation and characterization of DCspulsed with PAX3-FKHR peptide and the generation and characterization ofa CTL line specific for the PAX3-FKHR peptide and MHC molecule expressedon the DCs.

Example 3

This example demonstrates that the peptides of the present inventionstimulate T cells to kill tumor cells.

The human CTL line generated and characterized in Example 2 is testedfor the ability to kill human C1R-B7 target cells pulsed with thespecific RS10 peptide or a control SSI peptide from synovial sarcomathat also binds HLA-B7 (Worley et al., Cancer Res., 61, 6868-6875(2001)) (FIG. 1). In particular, specific cytotoxic activities aredetermined in a standard 4 h ⁵¹Cr release assay at variouseffector:target (E:T) ratios. Briefly, graded doses of viable effectorcells are plated in triplicate in 96-well U-bottom culture plates(Corning Glass, Corning, N.Y.) and co-cultured for 4 h with sodiumchromate-labeled (100 μCi; NEN, Boston, Mass.) peptide pulsed (10 μM)C1R-B7 target cells. In some experiments, the Rh5, RD and CTR tumor celllines are used as a target. Supernatants are collected, radioactivitymeasured, and specific lysis is calculated according to the equation:percentage of specific cytotoxicity=(experimental cpm−spontaneouscpm)/(maximum cpm−spontaneous cpm)×100.

Maximum ⁵¹Cr release is determined from supernatants of lysed targetcells incubated with Triton X-100 (5% v/v). Spontaneous release isdetermined from target cells incubated without added effector cells.

The lysis is clearly specific for the PAX-FKHR-derived RS10 peptide.Furthermore, the killing is restricted by the human HLA-B7 class I MHCmolecule as demonstrated by blockade of killing with antibody to HLA-B7(FIG. 2).

This example demonstrated that the generated CTL can lyse tumor cells.

Example 4

This example demonstrates that human CTL specific for the RS10 PAX-FKHRfusion peptide kill rhabdomyosarcoma cells expressing HLA-B7.

To determine whether this RS10 epitope is naturally processed andpresented by HLA-B7 in human tumor cells that express endogenousPAX-FKHR fusion protein, the lytic ability of the RS10-specific CTL lineis tested against a rhabdomyosarcoma tumor cell expressing HLA-B7 (Rh5),and two control lines RD and CTR not expressing HLA-B7 (FIG. 3). CTLactivity is assessed as described in Example 3.

Clear specific lysis of the Kh5 cells compared to the other controltumor cells indicates 1) that the RS10 epitope is indeed naturallyendogenously processed and presented in unmanipulated human tumor cells,and 2) that CTL raised against this epitope could kill human tumorcells.

This example demonstrated that human CTL specific for the RS10 peptidekill tumor cells expressing an MHC Class I molecule.

Example 5

This example demonstrates the generation and testing of variant peptidesof RS10.

To maximize immunogenicity, it is often helpful to modify the sequenceof an epitope to increase the affinity for the relevant MHC molecule, ina process that is called epitope enhancement (Berzofsky et al., Ann.N.Y. Acad. Sci., 690, 256-264 (1993), Berzofsky et al., Ann. N.Y. Acad.Sci., 754, 161-168 (1995), Ahlers et al., Proc. Natl. Acad. Sci USA, 94,10856-10861 (1997), Sarobe et al., J. Clin. Invest., 102, 1239-1248(1998), Ahlers et al., J. Clin. Invest., 108, 1677-1685 (2001),Berzofsky et al., Nature Reviews Immunology, 1, 209-219 (2001), Okazakiet al., J. Immunol., 171, 2548-2555 (2003), Oh et al., Cancer Research,64, 2610-2618 (2004), and Berzofsky et al., J. Clin. Invest., 113,1515-1525 (2004)). Such improvements in binding affinity can sometimesbe achieved by replacement of an amino acid residue causing an adverseinteraction with one that has a small, neutral side chain, such asalanine (Berzofsky et al., Ann. N. Y. Acad. Sci., 690, 256-264 (1993),Berzofsky et al., Ann. N. Y. Acad. Sci., 754, 161-168 (1995), Ahlers etal., Proc. Natl. Acad. Sci USA, 94, 10856-10861 (1997), Ahlers et al.,J. Clin. Invest., 108, 1677-1685 (2001), and Boehncke et al., J.Immunol., 150, 331-341 (1993)). To screen for such a possibility, aseries of peptides with Ala substitutions at each position in RS10 issynthesized and tested for binding to HLA-B7 in a T2-B7 binding assay.The variants with the Ala substitution at positions 3, 5, and 6(RS10-3A, RS10-5A, and RS10-6A, respectively) show higher bindingaffinity for HLA-B7 than the natural RS10 peptide (FIGS. 4 and 6). Theconcentration required for a 50% increase in HLA-B7 expression isdecreased from approximately 0.3 μM for RS10 to approximately 0.05 μMfor the RS10-3A variant.

To be sure that the epitope enhanced peptide with increased bindingaffinity for the HLA molecule has not simultaneously lost recognition bythe T cell, the ability of the RS10-specific human CTL to kill C1R-B7targets pulsed with the RS10-3A peptide is tested as described above. Asshown in FIG. 5, the specific killing observed confirms that theenhanced epitope is not so altered in the surface presented to the Tcell receptor that it had lost recognition by the human CTL. Thisenhanced peptide may therefore serve as a candidate vaccine to elicitspecific CTL immunity in HLA-B7⁺ patients with alveolarrhabdomyosarcoma, without the concern that self tolerance could dampenthe response, or that the tumor could escape by losing the fusionprotein.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An immunogenic peptide comprising SEQ IDNO: 3 with an alanine substitution at the third, fifth, or sixthposition of SEQ ID NO: 3, a functional portion thereof, or apharmaceutically acceptable salt thereof, wherein the peptide orfunctional portion thereof binds to an MHC Class I molecule.
 2. A fusionprotein comprising the immunogenic peptide of claim 1 and an MHC Class Imolecule, or a functional portion thereof.
 3. A conjugate comprising theimmunogenic peptide of claim
 1. 4. A pharmaceutical compositioncomprising the peptide of claim 1 and a pharmaceutically acceptablecarrier.
 5. A pharmaceutical composition comprising the fusion proteinof claim 2 and a pharmaceutically acceptable carrier.
 6. Apharmaceutical composition comprising the conjugate of claim 3 and apharmaceutically acceptable carrier.
 7. The immunogenic peptide of claim1, wherein the MHC Class I molecule is HLA-B7.
 8. The immunogenicpeptide of claim 1, wherein the peptide consists of 8 to 10 amino acids.9. The immunogenic peptide of claim 1, wherein the immunogenic peptidestimulates cytotoxic T lymphocytes.
 10. The immunogenic peptide of claim9, wherein the immunogenic peptide stimulates cytotoxic T lymphocytes tokill tumor cells in vitro.
 11. The immunogenic peptide of claim 10,wherein the tumor cells are alveolar rhabdomyosarcoma cells.